Messenger RNA (mRNA) directs protein synthesis. Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
The naturally occurring events that provide the disruption of the nucleic acid function, discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (Miller, et al., Anti-Cancer Drug Design 1987, 2, 117) and .alpha.-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2'-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research reagents and potential therapeutic purposes. At least for therapeutic purposes, the antisense oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express activity. One method for increasing membrane or cellular transport is by the attachment of a pendant lipophilic group.
Ramirez, et al., J. Am. Chem. Soc. 1982, 104, 5483, introduced the phospholipid group 5'-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3' and 5' positions. Subsequently Shea, et al., Nuc. Acids Res. 1990, 18, 3777, disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5'-phosphate on the 5'-terminus of the oligonucleotide. Certain of the Shea, et. al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra, et al., Tetrahedron Letters 1987, 28, 3581.
In other work, a cholesteryl group was attached to the inter-nucleotide linkage between the first and second nucleotides (from the 3' terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further by Letsinger, et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553. The aromatic intercalating agent anthraquinone was attached to the 2' position of a sugar fragment of an oligonucleotide as reported by Yamana, et al., Bioconjugate Chem. 1990, 1, 319.
Lemairte, et al., Proc. Natl. Acad. Sci. USA 1986, 84, 648; and Leonetti, et al., Bioconjugate Chem. 1990, 1, 149. The 3' terminus of the oligonucleotides each include a 3'-terminal ribose sugar moiety. The poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of this terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0. In this instance the lysine residue was coupled to a 5' or 3' phosphate of the 5' or 3' terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3' terminus of an oligonucleotide to link a peptide to the oligonucleotide as is described by Corey, et al., Science 1987, 238, 1401;
Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; and Corey, et al., J. Am. Chem. Soc. 1989, 111, 8524.
Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe a linking reagent for attaching biotin to the 3'-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3'-Amine on. It is also commercially available under the name 3'-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991, 32, 879. A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5'-terminus of an oligonucleotide is 5'-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg, et al., Antisense Research and Development 1991, 1, 161 to link fluorescein to the 5'-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3'-terminus of an oligonucleotide. Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297 described linking acridine on the 3'-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis, et al., Tetrahedron Letters 1987, 28, 5199 report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3' hydroxyl group of the 3' terminal nucleotide of the oligonucleotide. Chollet, Nucleosides & Nucleotides 1990, 9, 957 attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5' terminal phosphate of an oligonucleotide. They then used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to the oligonucleotide.
An EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer, et al., Proc. Natl. Acad. Sci. USA 1985, 82, 968. Fluorescein has been linked to an oligonucleotide in the same manner as reported by Haralambidis, et al., Nucleic Acid Research 1987, 15, 4857 and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser, et al., J. Am. Chem. Soc. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Sproat, et al., Nucl. Acids Res. 1987, 15, 4837, have synthesized 5'-mercapto nucleosides and incorporated them into oligonucleotides. Several phosphoramidites and H-phosphonates have been reported for introduction of a 5'-thiol linker via a phosphate linkage (see, Mori, et. al., Nucleosides and Nucleotides, 1989, 8, 649; WO 89/02931 (Levenson, et al.) published Apr. 6, 1989; Sinha, et al., Nucl. Acids. Res. 1988, 16, 2659). The amidites provide means for attachment of a HS--(CH.sub.2).sub.n --O--P(.dbd.O)--O---linkage to the oligomer. Also, disulfide-protected mercapto alkanols have been used to yield phosphoramidites (available from Glen Research, Sterling, Va. and Clontech, Palo Alto, Calif.). The same mercapto alkanoles have been attached to controlled pore glass (CPG) to give solid supports which yield 3'-thiolated oligonucleotides having a phosphate or thiophosphate linkage between the linker and the oligonucleotide. In another approach, oligonucleotides having 5'-amino linkers have been converted into oligonucleotides having 5'-thiol linkers by treatment with dithiobis-(N-succinimidyl) propionate or N-succinimidyl-3-(2-pyridyldithio) propionate followed by dithiothreitol (DTT) (See, Bischoff, et al., Anal. Biochem. 1987, 164, 336 and Gaur, et al., Nucl. Acids Res. 1989, 17, 4404). Asseline, et al., Tetrahedron 1992, 48, 1233 and Fidanza, et al., J. Am. Chem. Soc. 1992, 114, 5509, have used either the terminal or internucleotide thiosphophate groups to attach pendant groups. Fidanza, et al., J. Org. Chem. 1992, 57, 2340, have used cystamine (H.sub.2 N--CH.sub.2 --CH.sub.2 --S--S--CH.sub.2 --CH.sub.2 --NH.sub.2) to oxidize internucleotide H-phosphonate and provide a --P--NH--CH.sub.2 --CH.sub.2 --SH tether. Zuckerman, et. al., Nucleic Acids Res. 1987, 15, 5305, have used a 3'-S-alkyl thiol linker in a thymine nucleoside and incorporated the nucleoside into oligonucleotides. Ferentz, et al., (J. Org. Chem. 1990, 55, 5931 and J. Am. Chem. Soc. 1991, 113, 4000) have shown methods of attaching --NH--(CH.sub.2).sub.n --SH (n=2,3) at the 4-position of cytosine and the 6-position of adenosine.
However, there still remains a need in the art for methods of synthesis for nucleosides and oligonucleosides bearing further thiol-containing species.